Hi everyone, and welcome to Biology Professor. Today, we're gonna go over
how to conduct a Most Probable Number test. So a Most Probable Number test,
or MPN is sometimes how the method is referred to as the NPN method,
this is a method that is used to count bacteria, to measure the amount of bacteria
in a sample. And it is ideal for estimating the number of microbes in very dilute samples.
So when we say dilute, very dilute samples, we mean samples where the bacteria are not
very concentrated. So we're not talking about lab plates or lab cultures where you can do a plate
count or an optical density. These are samples where there are much fewer bacteria present or at
least that's what you suspect. An example of this would be various kinds of water and food testing,
so for example, testing certain kinds of water sources for coliform bacteria.
Coliform bacteria are bacteria that can ferment lactose and we have a lot of coliform bacteria
in human feces and so if there's coliforms in water, that's typically indicative of fecal
contamination. So the way the MPN method works is it uses changes in either color of broth
or changes in turbidity, that's the cloudiness that you see when bacteria are growing in liquid
culture, so it looks for either color change or turbidity change to detect bacterial growth.
And then you look at how many tubes either changed color or have noted turbidity and you can count
those tubes and then interpret your results by comparing your results to a standard reference
table. And we'll go over an example of that in a second, but when you are using the standard
reference table, it's important that you realize that the reference table will give you a number,
a most probable number an MPN, and then an MPN is equal to 1 CFU. CFU is a common way of referring
to bacteria and it stands for "colony forming unit". And basically that just means one viable
cell. So, just to recap, this method gives you a number, a most probable number, for how many
bacteria are in a certain sample and that equates to one colony forming unit, which is the same
thing as saying one viable cell, so one living cell, one cell that's capable of reproducing. So
let's go through an example. First, you have your inoculation. And so in this example, we're looking
at a water sample. Perhaps it's from a pond, perhaps it's from a recreational swimming area,
perhaps it's a drinking water sample - we have some water sample here and then we have three sets
of five lactose broth tubes. And remember that in this example, we're looking for coliform bacteria
and coliform bacteria can ferment lactose. You could do this method with other kinds of tests
where you're looking for a different kind of color change or a different growth in turbidity
and different kinds of broth, depending on the type bacteria you're looking at. So here we have
our three sets of five lactose brought tubes and what we do is we take ten milliliters from the
drinking water sample and inoculate each of these five tubes - so ten milliliters added to each one,
then we take one milliliter of the water sample and add it to each of these tubes, and then we
take 0.1 milliliters of water from this sample and add it to each of these tubes. And so we're having
this tenfold decrease each time in the amount of water that's being added and so theoretically
in the amount of viable coliforms that are being added to these tubes. Then we incubate overnight
at 37 degrees. Typically, you want to incubate for like 24 hours for this particular method,
then it's time to read results and what that means is you're looking for the color change.
So when coliform bacteria are present in these broth tubes, they will ferment the lactose and
that results in a color change. And so, for this particular test, we have four positive tubes here.
So four of them showed a color change, one did not. We have three positive tubes here, three
showed a color change, two did not. And then where we were adding the smallest amount of the water
sample, we've just got one positive tube, so one changed color and four did not. And so that is our
results that we then want to come and compare in the reference table. So our pattern, if you will,
was 4-3-1 and so we look at our reference table. If you're a student, this is often in the back of
a textbook or in the back of like a lab manual. So you look for the pattern of 4-3-1 and this tells
you that if you see the 4-3-1 pattern, then the MPN, the most probable number, of coliforms in the
original sample was 33 per 100 mL. Now, if we had gotten different patterns up here, if it had been
4-4-1 or if it had been 5-2-1 - different patterns will result in a different most probable number,
but with this pattern of 4-3-1, the best answer statistically is that for every 100 milliliters
of that sample we have 33 coliform bacteria. The table also gives us a 95% confidence range,
and for the pattern 4-3-1, the 95% confidence range is 11 to 93, so the most probable number
is 11 to 90 per 100 milliliters and all this means is that whereas 33 is like the best guess,
the most sort of statistically supported guess, you can be 95% confident that,
in this water sample that gave this pattern, there is somewhere between 11 and 93 viable
coliform bacteria per 100 mL of this sample. So that is how you conduct an MPN test. If you want
to learn more about bacterial growth, you can check out my video on bacterial growth curves
and binary fission, but that's it for today. Thanks for watching Biology Professor! :)